P383: USING CF-DNA AS STARTING MATERIAL FOR MRD STUDIES BY NGS IMPROVES THE SENSITIVITY OF THE METHOD AGAINST CTCS.
Authors: N. Álvarez Sánchez-Redondo, S. Dorado, A. Martín, L. Rufián, V. Garrido, A. Giménez, L. Moreno, Y. Heredia, J. Martínez-López, S. Barrio, R. Ayala
Journal: HemaSphere (2022)

Background: The quantification of Minimal Residual Disease (MRD) has become a relevant marker as it can detect acute myeloid leukemia (AML) patients with a high risk of relapse. Although the reference MRD method in AML is to quantify tumor cells present in bone marrow (BM), the study of circulating tumor cells (CTC) in peripheral blood (PB) is also being incorporated into clinical protocols. However, cfDNA as an MRD biomarker in AML has not yet been validated. Keywords: AML, MRD, Liquid Biopsy, NGS. Aims: The purpose of the study is to evaluate the use of cfDNA by NGS test as a new method for MRD quantification in AML and compare it application with the use of PB CTCs in the follow-up of the disease. Methods: Three AML patients treated at the Hospital Universitario 12 de Octubre and with cfDNA available have been included. The mutational profile was defined by NGS (Ion Torrent System) at diagnosis using a panel of 42 genes involved in myeloid pathologies. Then, somatic mutations were selected, and MRD quantification was performed in liquid biopsy combining whole blood cells (WBC) and cfDNA. Both fractions were informatically differentiated by including molecular tags in the first PCR amplification. A total of 18 follow-up samples were analyzed with a threshold for EMR positivity set at 1x10^ (-4) as we previously described (Onecha E et al. Haematologica 2019). An average of 66ng of cfDNA and 660ng of gDNA from WBC were used. The results were compared with those obtained by multiparametric flow cytometry (MFC) in BM. Results: In the first patient, four variants affecting the genes CBL (VAF =7.4%), MPL (50%), JAK2 (51.5%) and ETV6 (10,6%) were detected at diagnosis in BM. The Leukemia-associated variant affecting ETV6 was selected as an MRD biomarker and detected in cfDNA (0.86%) post-consolidation 1. On this time-point (TP), the result of MRD by MFC on BM was also positive (0.002%), being negative in CTC-PB by NGS. The Non-leukemia-associated variants (JAK2, MPL, CBL) were also followed-up. Of interest, those alterations showed constant VAF levels in pre-transplant remission. After transplant, the VAF of JAK2 and MPL correlated with the donor chimerism studied by conventional methods after transplantation. On the other hand, CBL VAF was below the threshold sensitivity limit (0.01%) during all follow-ups. This result that CBL was also associated with leukemia, likely affecting a treatment-sensitive subclone. In patient 2, variants affecting IDH2 (92%), RUNX1 (43%), and DNMT3A (49%) genes were detected by at diagnosis. IDH2 mutation was selected as MRD biomarker, showed a VAF of 9.4% at relapse, correlating with the blast count of 10% defined by the morphology study of BM. Of note, in this TP, MRD-BM by MFC was 0.011%. In patient 3, the SF3B1 (43%) and EPAS1 (49%) mutations were detected by NGS in BM at diagnosis. The leukemia-associated variant (SF3B1) was observed in the cfDNA sample (0,0003) at the first follow-up sample but was negative at the following TPs. MRD by CTCs and MFC-BM were also negative. This patient has not relapsed to date (follow-up time six months after diagnosis). Image:Summary/Conclusion: MRD quantification based on the use of cfDNA by NGS offers promising results and, in the future, could be a good option for disease monitoring or early detection of relapse. The results of the whole series will be presented at the meeting.